The present invention, in some embodiments thereof, relates to therapy and, more particularly, but not exclusively, to compositions and methods utilizing aminoglycoside analogs in the treatment of Rett syndrome.
Rett syndrome (RTT, MIM 312750) is an X-linked postnatal neurodevelopmental disorder predominantly occurring in girls with a worldwide incidence of 1/10,000-15,000 female births. After normal development, during the first 6 to 18 months developmental stagnation and then regression occurs. During the phase of regression, purposeful hand use and language are lost while gross motor functions are relatively preserved. After the phase of regression the clinical picture remains stable for many years.
The major causative factor of RTT is deficiency of the X-linked methyl CpG-binding protein 2 (MeCP2) at Xq28, in which over 200 mutations have been identified so far in classical and atypical RTT patients. The majority of RTT causative mutations involve C.T transitions at the CpG hot-spots leading to missense, nonsense and frame-shift mutations, mostly originating de novo in the paternal germline. Phenotypic heterogeneity in RTT has been related, for the most part, to MECP2 mutation type and localization, as well as X chromosome inactivation (XCI) pattern.
The MECP2 gene encodes two isoform proteins, MeCP2_e1 and MeCP2_e2 products of an alternative initiation at exon 1 and splicing of exon 2, both of which are nuclear and co-localize with the methylated heterochromatin. Studies have shown that MeCP2 role in neurons is flexible and complex, as MeCP2 has been implicated in both repression and activation of a large number of genes, in modulation of RNA splicing, and has been suggested to affect global chromatin structure impacting on the entire neuronal genome.
Studies conducted with RTT mouse models showed that MeCP2 dysfunction in mature neurons accounts for RTT symptoms and that postnatal restoration of MeCP2 deficiency in the CNS, even after RTT onset, can lead to the reversal of neurological symptoms. These findings have led to the notion that RTT rescue may be achieved by pharmacological treatment that may induce MeCP2 up-regulation in MeCP2 deficient neurons, nonetheless considering the importance of correct MeCP2 dosage.
Significant proportion (up to 40%) of the classical RTT is caused by MECP2 nonsense mutations, leading to premature translational termination and truncated protein products. Studies using recombinant MeCP2 constructs harboring the most common RTT nonsense mutations, R168X, R255X, R270X and R294X, showed that gentamicin and geneticin can recover MeCP2 read-through efficiency up to 10-22% depending on the nucleotide context of a nonsense mutation [Brendel et al. (2009) Pediatr Res 65:520-523; Brendel et al., J Mol Med (2011) 89:389-398]. In addition, the recovered MeCP2 protein was traced to the cell nucleus suggesting that gentamicin does not interfere with its nuclear localization.
However, clinical applicability of aminoglycosides of the gentamicin family has been compromised by parallel findings of significant toxicity associated with its long-term administration and with reduced suppression efficiency at subtoxic doses [Kerem E (2004) Curr Opin Pulm Med 10: 547-552], in addition to its limited permeability through the blood-brain-barrier [Keeling K M, Bedwell D M (2005) Current Pharmacogenomics 3:259-269].

Some recently developed synthetic aminoglycosides have demonstrated significantly improved effects compared to gentamicin, evident in substantially higher suppression in RTT model and reduced acute toxicity in vitro [Kandasamy et al. J Med Chem 2012; 55:10630-43; Vecsler et al. PLoS One 2011; 6:e20733].
WO 2007/113841, by some of the present inventors, which is incorporated by reference as if fully set forth herein, teaches a class of paromomycin-derived aminoglycosides, which were designed specifically to exhibit high premature stop-codon mutations readthrough activity while exerting low cytotoxicity in mammalian cells and low antimicrobial activity, and can thus be used in the treatment of genetic diseases. This class of paromomycin-derived aminoglycosides was designed by introducing certain manipulations of a paromamine core, which lead to enhanced readthrough activity and reduced toxicity and antimicrobial activity. The manipulations were made on several positions of the paromamine core.

One such manipulation of the paromamine core which has been described in WO 2007/113841 is the determination of the beneficial role of a hydroxyl group at position 6′ of the aminoglycoside core (see, for example, NB30 and NB54 below).

Another manipulation of the paromamine core which has been defined and demonstrated in WO 2007/113841 is the introduction of one or more monosaccharide moieties or an oligosaccharide moiety at position 3′, 5 and/or 6 of the aminoglycoside core. This manipulation is reflected as “Ring III” in the exemplary compounds NB30 and NB54 shown hereinabove.
An additional manipulation of the paromamine core which has been defined and demonstrated in WO 2007/113841 is the introduction of an (S)-4-amino-2-hydroxybutyryl (AHB) moiety at position 1 of the paromamine core. This manipulation is reflected in exemplary compound NB54 shown hereinabove. It has been demonstrated that such an introduction of an AHB moiety provides for enhanced readthrough activity and reduced toxicity.
An additional manipulation of the paromamine core which has been described in WO 2007/113841 is the substitution of hydrogen at position 6′ by an alkyl such as a methyl substituent. This manipulation has been exemplified in a derivative of compounds NB30 and NB54, referred to as NB74 and NB84 respectively.

Vecsler et al. [(2011) PLoS ONE 6(6): e20733] have demonstrated that one of the compounds disclosed in WO 2007/113841 (NB54) induce dose-dependent suppression of MECP2 nonsense mutations more efficiently than gentamicin, which was evident at concentrations as low as 50 mg/ml. The read-through activity was mutation specific, with maximal full-length MeCP2 recovery in R168X (38%), R270X (27%) and R294X (18%). In addition, the recovered MeCP2 was translocated to the cell nucleus and led to parallel increase in one of the most important MeCP2 downstream effectors, the brain derived neurotrophic factor (BDNF).
Brendel et al (2011), supra, describe studies conducted in a mouse model carrying the R168X mutation in the MECP2 gene. Transfected HeLa cells expressing mutated MeCP2 fusion proteins and mouse ear fibroblasts isolated from the mouse model were treated with gentamicin and some of the aminoglycoside analogs described in WO 2007/113841 (NB30, NB54 and NB84). Readthrough of the R168X mutation in mouse ear fibroblasts using gentamicin was detected but at lower level than in HeLa cells, and the readthrough product, full-length Mecp2 protein, was located in the nucleus. NB54 and NB84 induced readthrough more effectively than gentamicin, with the readthrough of nonsense mutations achieved not only in transfected HeLa cells but also in fibroblasts of the generated Mecp2R168X mouse model.
International Patent Application Publication No. WO 2012/066546, by some of the present inventors, which is incorporated by reference as if fully set forth herein, teaches additional manipulations of the paromamine core, resulting in pseudo-trisaccharide aminoglycosides, characterized by a core structure based on Rings I, II and III of paromomycin with the addition of an alkyl in position 5″ on Ring III. The chemical structures of exemplary aminoglycosides disclosed in WO 2012/066546 are presented in Background Art FIG. 1.